The premise of this application is the following: Understanding the fundamental mechanisms that control the switching of globin gene expression during development and maintain silencing of fetal hemoglobin (HbF) in the adult is essential as a foundation on which to design targeted therapy for the major hemoglobin disorders-- sickle cell anemia and -thalassemia. Recent findings establish BCL11A as a major transcriptional repressor that restricts mouse embryonic -like globin expression to the primitive erythroid lineage and silences human -globin expression in mice (harboring a transgene with the entire human -globin locus). Pan-hematopoietic or erythroid-specific inactivation of BCL11A alone leads to substantial reactivation of HbF expression, and prevents disease in humanized, genetically engineered mice with sickle cell disease (SCD). Thus, the aims of the current application focus specifically on how BCL11A functions to regulate globin switches, as an improved understanding will provide critical insights into potential strategies to target down-regulation or inhibition of BCL11A expression or function to reactivate ?-globin expression in patients with the major hemoglobin disorders. In order to move forward in the discovery of small molecules that impair the function of BCL11A or its critical partner proteins, we believe it is necessary to define in depth the manner in which BCL11A functions in globin repression by delineating its principal functional domains. To this end, we will employ a BCL11A null adult erythroid mouse erythroleukemia cell line created by genome engineering to determine the domains of the protein necessary for globin repression. Second, we will determine how these domains interact with other proteins; participate in protein multimerization, and/or DNA binding. In parallel, we will explore the potential to humanize mice to express BCL11A in a human rather than a mouse pattern in development in an effort to generate an improved animal model for globin gene switching. A model of this nature would facilitate testing of gene manipulations and small molecules that reactivate HbF, and ultimately contribute to the development of new therapeutics for the major hemoglobin disorders.